The emergence of antibiotic resistance among different pathogenic bacteria is a major global problem. Within the last two decades, antibiotic-resistant strains emerged at an alarming rate;yet, only a few new antibiotics were developed. The gram-negative opportunistic pathogen Pseudomonas aeruginosa is an example of this problem. Multidrug resistant clones of P. aeruginosa may persist within the hospital environment for several years by transferring among patients. As a result, research efforts are directed at developing alternative P. aeruginosa therapies that would weaken the bacteria but would not induce the production of resistant mutants. One target for these therapies is the cell-to-cell communication system, or quorum sensing (QS) system, through which P. aeruginosa synchronizes the production of numerous virulence factors including exotoxins. This occurs through secondary metabolites or small communication molecules termed autoinducers. Due to their importance, P. aeruginosa carries multiple autoinducers. Therefore, identifying and fully characterizing these autoinducers is essential for designing future therapies that block their actions. At present, three P. aeruginosa autoinducers have been fully characterized: 3OC12-HSL, C4-HSL, and PQS - the Pseudomonas quinolone signal. Recently, we and others have identified pseudoverdine, a novel quinolone molecule which is synthesized from a precursor molecule termed paerucumarin. These molecules are synthesized by the pvc operon which is located adjacent to ptxR. The ptxR gene codes for the P. aeruginosa global regulator PtxR, which regulates the expression of numerous P. aeruginosa virulence genes. Evidence suggests that pseudoverdine and/or paerucumarin activates PtxR. Additional analysis, using the murine model of systemic infection, revealed that specific deletions within the pvc operon reduced the in vivo virulence of P. aeruginosa significantly. We have synthesized paerucumarin and we are currently synthesizing pseudoverdine. We hypothesize that pseudoverdine is a novel communication molecule that P. aeruginosa utilizes to coordinate the production of different virulence factors. The specific aims of the application are: 1) to examine the influence of synthesized pseudoverdine and/or paerucumarin on the production of virulence factors as well as the expression of different P. aeruginosa genes, 2) to determine if pseudoverdine and/or paerucumarin enhance PtxR binding to its target genes, and 3) to determine the effect of exogenously added pseudoverdine and/or paerucumarin on the in vivo virulence of P. aeruginosa using the murine model of systemic infection. These experiments will be done using a specific assay for each virulence factor examined, quantitative RT-PCR, transcriptional fusion studies, and microarray experiments. Additionally, we will determine if pseudoverdine and/or paerucumarin induce a conformational change in PtxR using DNA/gel shift assays and other assays to detect conformational changes. We will examine the effect of these molecules on the spread P. aeruginosa both locally within the infected tissues and systemically within the blood and internal organs of thermally-injured mice. In addition, we will assess the effect of the molecules themselves on the host immune response by measuring the level of expression of pro- inflammatory cytokines in response to subcutaneous injection of pseudoverdine and/or paerucumarin. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a microbe that produces serious illness in people with weakened resistance, such as severely burned patients and patients with lung infections. The organism causes this harmful effect by producing numerous factors that damage the host. This research is designed to study a system that controls production of these factors. The final aim from learning this is to stop the organism from producing such factors.